Circadian Clock In Mice Research Articles

Nobiletin Stimulates Adrenal Hormones and Modulates the Circadian Clock in Mice.

Polymethoxyflavonoids, such as nobiletin (abundant in Citrus depressa), have been reported to have antioxidant, anti-inflammatory, anticancer, and anti-dementia effects, and are also a circadian clock modulator through retinoic acid receptor-related orphan receptor (ROR) α/γ. However, the optimal timing of nobiletin intake has not yet been determined. Here, we explored the time-dependent treatment effects of nobiletin and a possible novel mechanistic idea for nobiletin-induced circadian clock regulation in mice. In vivo imaging showed that the PER2::LUC rhythm in the peripheral organs was altered in accordance with the timing of nobiletin administration (100 mg/kg). Administration at ZT4 (middle of the light period) caused an advance in the peripheral clock, whereas administration at ZT16 (middle of the dark period) caused an increase in amplitude. In addition, the intraperitoneal injection of nobiletin significantly and potently stimulated corticosterone and adrenaline secretion and caused an increase in Per1 expression in the peripheral tissues. Nobiletin inhibited phosphodiesterase (PDE) 4A1A, 4B1, and 10A2. Nobiletin or rolipram (PDE4 inhibitor) injection, but not SR1078 (RORα/γ agonist), caused acute Per1 expression in the peripheral tissues. Thus, the present study demonstrated a novel function of nobiletin and the regulation of the peripheral circadian clock.

Abstract P2-02-07: Patients’ selection of daily timing of oral intake of adjuvant hormonotherapy (HT) and everolimus (EVE) for high risk early breast cancer in the UCBG-UNIRAD phase III trial

Abstract Background: Everolimus (EVE) addition to adjuvant hormonotherapy (HT) for high-risk early breast cancer (BC) did not improve 3-year disease-free survival (DFS) compared with ET alone in the randomized UNIRAD trial (NCT01805271) (1). Most patients (pts) withdrew from EVE for adverse events nearly midway before the expected treatment duration of 2 years. The main EVE target involves m-TOR-induced S6 protein phosphorylation, which is controlled by the molecular circadian clock in mice and cells. Toward the analysis of possible circadian time-dependencies in EVE toxicities and efficacy, we first determine the actual distribution of the daily times chosen by the patients for both oral EVE and HT intakes. Patients and methods: The registered pts were asked to record the clock hours they chose for both HT and EVE or placebo (PLAC) intakes within four possible daily 6-h slots, i.e. from 06:00 to 11:59 (morning), 12:00 to 17:59 (midtime), 18:00 to 23:59 (evening), or 24:00 to 05:59, (night) in a daily diary throughout their participation in the trial. Modifications in times of drugs intake were reported. Comparisons between groups involved Kruskal-Wallis sum test and Pearson’s Chi-squared and Fisher’s exact tests. Results: Out of 1,278 randomized patients, 1063 (83,1 %) recorded the times of EVE or PLAC intakes, 852 recorded those for HT intakes. Only 10 pts reported night EVE/PLAC intakes and were not considered here. Of the 1053 evaluated pts; 549 pts took EVE/PLAC in the morning, 82 pts at midtime and 422 in the evening. As compared with evening intakes, morning or midtime intakes of EVE or PLAC were significantly associated with an older age, and post-menopausal status, whilst younger and premenopausal women preferentially chose evening intakes (p< 0.001 for both). Consistently, HT with aromatase inhibitors (AI) were mostly taken in the morning or at midtime, whereas tamoxifene (TAM) was mostly taken the evening (p = 0.001). Tumor size, lymph nodes involvement, histological grade, hormonal receptors status, and EVE initial dose were similarly distributed among the three time slots (Table 1). A similar distribution of oral intake times as in the whole timing study population was found according to age, menopausal status and HT type in the EVE arm (N=508 pts), and in the HT population (+/- EVE or PLAC) (N=852 pts). Initial oral timing intake was modified for EVE by 50 of 508 pts (10%) and for HT by 10 of 852 pts (1%). Conclusion: To the best of our knowledge, it is the first time that the patients’ spontaneous selection of daily times for oral intakes of a targeted agent (EVE) and HT is reported in a large series of early breast cancer pts. Age and menopausal status were important determinants of patient-selected daily timing intakes of both EVE and HT, as well as HT types. These findings will be carefully considered in the analyses of possible EVE and HT timing effects on adverse events and efficacy of the current adjuvant regimens. 1) Bachelot T et al, J Clin Oncol. 2022 May 23 Citation Format: Sylvie Giacchetti, Anne-Sophie Hamy, Thomas Bachelot, Jérôme Lemonnier, Fabrice Andre, David A. Cameron, Judith Bliss, Sylvie Chabaud, Anne-Claire Hardy-Bessard, Magali Lacroix-Triki, Jean-Luc Canon, Hervé R. Bonnefoi, Mario Campone, Paul Cottu, Florence Dalenc, Annabelle Ballesta, Francis Lévi, Enora Laas. Patients’ selection of daily timing of oral intake of adjuvant hormonotherapy (HT) and everolimus (EVE) for high risk early breast cancer in the UCBG-UNIRAD phase III trial. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-02-07.

The Effect of Diet on the Cardiac Circadian Clock in Mice: A Systematic Review.

Circadian rhythms play important roles in regulating physiological and behavioral processes. These are adjusted by environmental cues, such as diet, which acts by synchronizing or attenuating the circadian rhythms of peripheral clocks, such as the liver, intestine, pancreas, white and brown adipose tissue, lungs, kidneys, as well as the heart. Some studies point to the influence of diet composition, feeding timing, and dietary restriction on metabolic homeostasis and circadian rhythms at various levels. Therefore, this systematic review aimed to discuss studies addressing the effect of diet on the heart clock in animal models and, additionally, the chronodisruption of the clock and its relation to the development of cardiovascular disorders in the last 15 years. A search was conducted in the PubMed, Scopus, and Embase databases. The PRISMA guide was used to construct the article. Nineteen studies met all inclusion and exclusion criteria. In summary, these studies have linked the circadian clock to cardiovascular health and suggested that maintaining a robust circadian system may reduce the risks of cardiometabolic and cardiovascular diseases. The effect of time-of-day-dependent eating on the modulation of circadian rhythms of the cardiac clock and energy homeostasis is notable, among its deleterious effects predominantly in the sleep (light) phase and/or at the end of the active phase.

Chronic Treatment with Metformin Has No Disrupting Effect on the Hepatic Circadian Clock in Mice.

Background and Objectives: The antidiabetic agent metformin is known to activate AMP-activated protein kinase (AMPK) in various tissues. Because AMPK can modulate intracellular circadian clocks through regulating the stability of clock components, a single dose of metformin has been reported to affect circadian clocks in the peripheral tissues. In this study, therefore, we investigated whether chronic treatment with metformin causes the impairment of circadian clocks, especially if given at an inappropriate time. Materials and Methods: Non-diabetic C57BL/6J mice were allowed access to food only during 4 h at the beginning of the dark period, and repeatedly i.p. injected with a nearly maximum non-toxic dose of metformin, once daily either at 4 h after the beginning of the dark period or at the beginning of the light period. Diabetic ob/ob mice were given free access to food and treated with metformin in drinking water. Results: Under the controlled feeding regimen, 8-day treatment with metformin did not alter the mRNA expression rhythms of clock genes in both liver and adipose tissue of C57BL/6J mice, regardless of dosing time. In addition, chronic treatment with metformin for 2 weeks affected hepatic AMPK activation rhythm but did not disrupt the circadian clocks in the liver and adipose tissues of the ob/ob mice. Conclusions: These results mitigate concerns that treatment with metformin impairs peripheral circadian clocks, although confirmation is needed in humans.

REG\u03b3 regulates circadian clock by modulating BMAL1 protein stability

Endogenous clocks generate rhythms in gene expression, which facilitates the organisms to cope through periodic environmental variations in accordance with 24-h light/dark time. A core question that needs to be elucidated is how such rhythms proliferate throughout the cells and regulate the dynamic physiology. In this study, we demonstrate the role of REGγ as a new regulator of circadian clock in mice, primary MEF, and SY5Y cells. Assessment of circadian conduct reveals a difference in circadian period, wheel mode, and the ability to acclimate the external light stimulus between WT and KO littermates. Compared to WT mice, REGγ KO mice attain the phase delay behavior upon light shock at early night. During the variation of 12/12 h light/dark (LD) exposure, levels of Per1, Per2, Cry1, Clock, Bmal1, and Rorα circadian genes in suprachiasmatic nucleus are significantly higher in REGγ KO than in WT mice, concomitant with remarkable changes in BMAL1 and PER2 proteins. In cultured cells depleted of REGγ, serum shock induces early response of the circadian genes Per1 and Per2 with the cyclic rhythm maintained. Mechanistic study indicates that REGγ directly degrades BMAL1 by the non-canonical proteasome pathway independent of ATP and ubiquitin. Silencing BMAL1 abrogates the changes in circadian genes in REGγ-deficient cells. However, inhibition of GSK-3β, a known promoter for degradation of BMAL1, exacerbates the action of REGγ depletion. In conclusion, our findings define REGγ as a new factor, which functions as a rheostat of circadian rhythms to mitigate the levels of Per1 and Per2 via proteasome-dependent degradation of BMAL1.

341 Age and circadian regulation of cutaneous innate antimicrobial immunity

Neonates and elderly are at increased risk for skin viral and bacterial infections that have the potential to cause systemic malaise and death. Notably, under healthy conditions, and predominantly previously studied in adult skin, a natural antimicrobial defense program protects the host against microbial infections. Interestingly, newborns and elderly have immature diurnal circadian rhythms. Unknown to us is whether age and/or circadian rhythm regulate innate antimicrobial immunity in the skin. Here, we aimed to address the question whether an immature perinatal and dysregulated circadian clock suppresses innate antimicrobial peptide and protein expression, which consequently allows for predisposition to infections. We hypothesized that age-related maturation of the daily circadian rhythm via CLOCK/BMAL1 facilitates transcriptional regulation of innate antimicrobial host defense molecules in the skin. Innate antimicrobial peptides and proteins (AMPs), including antiviral proteins, play a pivotal role in innate immune defense system to protect against invading pathogens. AMPs are expressed in most barrier tissues, including the skin. Here, we show that many cutaneous AMP mRNAs are not expressed at birth, but their expression induction instead coincide with maturation of the circadian clock in mice. We also show using ex vivo studies in adult human skin that homeostatic AMP mRNA expression changes with age. Our findings indicate that AMP expression varies with age in human and murine skin, possibly in relation to altered expression of CLOCK/BMAL1.

Non-invasive 40-Hz Light Flicker Ameliorates Alzheimer's-Associated Rhythm Disorder via Regulating Central Circadian Clock in Mice.

Alzheimer’s disease (AD) patients often exhibit perturbed circadian rhythm with fragmented sleep before disease onset. This study was designed to evaluate the effect of a 40-Hz light flicker on circadian rhythm in an AD mouse model (APP/PS1). Locomotor rhythms recordings were conducted to examine the circadian clock rhythm in APP/PS1 mice. Molecular biology analyses, including western blot and real-time qPCR assays, were conducted to assess the changes in circadian locomotor output cycles kaput (CLOCK), brain and muscle arnt-like protein-1 (BMAL1), and period 2 (PER2). In addition to determining the direct effect of a 40-Hz light flicker on hypothalamic central clock, whole-cell voltage-clamp electrophysiology was employed to record individual neurons of suprachiasmatic nucleus (SCN) sections. The results reported herein demonstrate that a 40-Hz light flicker relieves circadian rhythm disorders in APP/PS1 mice and returns the expression levels of key players in the central circadian clock, including Clock, Bmal1, and Per2, to baseline. Moreover, the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in SCN neurons is significantly lower in APP/PS1 mice than in the control, and the amplitude of sIPSCs is decreased. Exposure to a 40-Hz light flicker significantly increases the sIPSC frequency in SCN neurons of APP/PS1 mice, with little effect on the amplitude. However, the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) are both unaffected by a 40-Hz light flicker. The data suggest that a 40-Hz light flicker can ameliorate AD-associated circadian rhythm disorders, presenting a new type of therapeutic treatment for rhythm disorders caused by AD.

Angptl8 mediates food-driven resetting of hepatic circadian clock in mice

Diurnal light-dark cycle resets the master clock, while timed food intake is another potent synchronizer of peripheral clocks in mammals. As the largest metabolic organ, the liver sensitively responds to the food signals and secretes hepatokines, leading to the robust regulation of metabolic and clock processes. However, it remains unknown which hepatokine mediates the food-driven resetting of the liver clock independent of the master clock. Here, we identify Angptl8 as a hepatokine that resets diurnal rhythms of hepatic clock and metabolic genes in mice. Mechanistically, the resetting function of Angptl8 is dependent on the signal relay of the membrane receptor PirB, phosphorylation of kinases and transcriptional factors, and consequently transient activation of the central clock gene Per1. Importantly, inhibition of Angptl8 signaling partially blocks food-entrained resetting of liver clock in mice. We have thus identified Angptl8 as a key regulator of the liver clock in response to food.

Ablation of Sirtuin 1 Deacetylase Activity Induces Pulmonary Emphysema by Inducing Cellular Senescence and Disrupting Circadian Clock in Mice (P01-022-19)

ObjectivesSirtuin 1 (SIRT1), a NAD+-dependent protein/histone deacetylase has the capability to extend life span, delay aging, and prevent aging-related diseases. There are several reports showing that there is no significant decline in SIRT1 protein with age, indicating that SIRT1 protein levels alone may not reflect its deacetylase activity. We investigated the causal effect of systemic ablation of SIRT1 deacetylase activity on aging-related pulmonary disease development in mice. MethodsWe used Sirt1y/y homozygous male mice carrying a point mutation (H355Y) that ablates the deacetylase activity, along with their wild type littermates (Sirt1+/+), and followed them for 6, 10 and 18 months of age. ResultsSirt1y/y homozygous mice developed severe pulmonary emphysema at the ages of 6, 10 and 18 months, with the respective incidences of 33%, 100% and 100%, while the Sirt1+/+wild-type mice only developed emphysema (13% incidence) at 18 months of age. The development of emphysema in Sirt1y/y mice was accompanied with higher protein levels of matrix metalloproteinase (MMP)-2, MMP9, and tissue inhibitor of metalloproteinase-1, and ratio of cleaved/total anti-poly (ADP-ribose) polymerase. The ablation of SIRT1 activity significantly up-regulated mRNA expression of hypoxia-inducible factor-1α and retinoic acid receptor-b, while p21 protein and phosphorylated AMPK increased and phosphorylated ribosomal S6 decreased, suggesting the association of ablation of SIRT1 activity with cellular quiescence and senescence. Additionally, the lack of SIRT1 activity down-regulated the mRNA expression of circadian clock genes (BMAL1, NPAS2, CRY1, CRY2) in the lungs of Sirt1y/y mice, as compared with that of Sirt1+/+ mice. There were no inflammatory responses in the lungs (e.g., inflammatory cell infiltrations, mRNA expressions of IL-6 and TNFα) in Sirt1y/y homozygous mice compared to Sirt1+/+ mice. ConclusionsThe lacking of SIRT1 enzymatic activity plays a major role in the susceptibility of organs to aging and pulmonary emphysema development by inducing cellular senescence and disrupting circadian clock genes. Funding SourcesUSDA/ARS (58-1950-0074) and NIFA/AFRI (2017-67017-26363).

Rhythmic Food Intake Drives Rhythmic Gene Expression More Potently than the Hepatic Circadian Clock in Mice

Every mammalian tissue exhibits daily rhythms in gene expression to control the activation of tissue-specific processes at the most appropriate time of the day. Much of this rhythmic expression is thought to be driven cell autonomously by molecular circadian clocks present throughout the body. By manipulating the daily rhythm of food intake in the mouse, we here show that more than 70% of the cycling mouse liver transcriptome loses rhythmicity under arrhythmic feeding. Remarkably, core clock genes are not among the 70% of genes losing rhythmic expression, and their expression continues to exhibit normal oscillations in arrhythmically fed mice. Manipulation of rhythmic food intake also altersthe timing of key signaling and metabolic pathways without altering the hepatic clock oscillations. Our findings thus demonstrate that systemic signals driven by rhythmic food intake significantly contribute to driving rhythms in liver gene expression and metabolic functions independently of the cell-autonomous hepatic clock.

Rhythmic Food Intake Drives Rhythmic Gene Expression More Potently than the Hepatic Circadian Clock in Mice

Every mammalian tissue exhibits daily rhythms in the expression of thousands of genes to control the activation of tissue-specific processes at the most appropriate time of the day. Much of this rhythmic expression is thought to be driven cell-autonomously by molecular circadian clocks present throughout the body. By manipulating the daily rhythm of food intake in the mouse, we here show that more than 70% of the cycling mouse liver transcriptome loses rhythmicity under arrhythmic feeding. Remarkably, this loss of rhythmic gene expression is independent of the hepatic circadian clock, which continues to exhibit normal oscillations in core clock gene expression. Manipulation of rhythmic food intake also alters the timing of key signaling and metabolic pathways without altering the hepatic clock oscillations. Our findings thus demonstrate that systemic signals driven by rhythmic food intake significantly contribute to driving rhythms in liver gene expression and metabolic functions independently of the cell-autonomous hepatic clock.

From the Literature

From the Literature

FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice

The circadian clock generates biological rhythms ofmetabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk), an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient dietaltered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis). We conclude that light-independent mechanisms of FAD regulate CRY and contribute toproper circadian oscillation of metabolic genes in mammals.

Abstract P4-06-06: Consistent dosing-time dependent tolerability of everolimus (EV) in a pilot study in women with metastatic breast cancers (MBC) and in a mouse chronopharmacology investigation

Abstract Background The molecular circadian clock regulates mTOR activity, the main target of EV. EV use is hampered by adverse events in MBC patients (pts). Knowing the optimal timing of EV oral intake could improve tolerability, thus compliance and treatment efficacy. Objective: To assess the relevance of EV timing for side effects in MBC pts, and to determine the impact of EV timing for body weight, circadian rest-activity pattern, and liver circadian clock in mice. Materials and methods: From 2013 to 2014, 19 MBC pts were treated with EV 10mg plus hormonotherapy at St Louis hospital Breast Cancer unit. Morning or Evening oral intake was prescribed by the medical oncologist in charge of each patient for both EV and hormonotherapy. Side effects were compared according to EV timing. We further investigated whether oral (po) EV timing mattered mice regarding tolerability. EV (5-20 mg/kg/day x 14 days) was administered through oral gavage at 4 circadian times, 6 hours apart to different groups of synchronized male or female B6D2F1 mice (n=32/sex). Body weight change and lethal toxicity were the endpoints. EV effects were further determined on clock gene Per2 expression in the liver of Per2::luciferase (Per2::luc) mice. Per2::luc expression was monitored in freely-moving mice every minute for 3 days before, 5 days during and 3 days after daily EV at one of two timepoints using Real Time-Biolumicorder (RT-BIO, Geneva, Sw). Periods and amplitudes of Per2::luc rhythms were determined with spectral analysis. Results: Nine pts took EV in the morning and 10 in the evening for a median duration of 7.5 months (1.3-19.2 m). Median age was 59 (39-85), median delay between metastatic disease and EV treatment was 52.5 months. Pts had a median of 3 prior lines of treatment for MBC. EV was associated with exemestane (13 pts), or tamoxifen (6 pts). Evening EV was associated with higher occurrence of edema (6 vs 2 pts), fatigue (9 vs 2 pts), and pneumonia (3 vs 0 pts). Equitoxic doses of EV were twice as high in female as compared to male mice. EV timing at the beginning of the light (rest) span resulted in a lethal toxicity rate of 25% in female mice receiving 20 mg/kg/d and 50% in male mice dosed with 10 mg/kg/d). In contrast all the mice treated with EV at the beginning of the dark (activity) span survived, irrespective of dose level. Maximum body weight loss ranged from 5% following EV timing near activity onset to 12% after EV delivery near rest onset, both in males (5 mg/kg/d) and females (10 mg/kg/d) (ANOVA p<0.001). The circadian expression of liver Per2::luc was abolished following EV dosing near rest onset. Per2::luc remained rhythmic in mice receiving EV at the beginning of the activity span. Conclusion: Tolerability was largely improved by the delivery of EV near the onset of the activity span both in women with MBC and in male or female mice. Morning administration of EV could minimize metabolic alterations and fatigue in humans. This timing should further avoid circadian disruption in the liver molecular clocks thus ensure better treatments tolerability. Citation Format: Giacchetti S, Li XM, Ozturk N, Cuvier C, Machowiak J, Arrondeau J, Chang-Marchand Y, Espié M, Okyar A, Lévi F. Consistent dosing-time dependent tolerability of everolimus (EV) in a pilot study in women with metastatic breast cancers (MBC) and in a mouse chronopharmacology investigation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-06.

Risperidone resets the circadian clock in mice

Risperidone is an atypical antipsychotic that is active at multiple dopamine and serotonin receptor subtypes. Based on its high affinity for serotonin receptors, we predicted that it might reset circadian rhythms in a nocturnal rodent. We report temporally differentiated and differential effects of various doses of risperidone on the voluntary locomotor activity rhythm in the Indian field mice, Mus booduga. Risperidone (0.5 mg/kg) elicited phase delays at phases between CT (circadian time) 12 to CT18 and CT0 to CT3, and phase advances at CT6, CT9 and CT21. However, mice injected at CT6 showed maximum advances (1.299 ± 0.286 h), whereas at CT15 showed maximum delays (−1.514 ± 0.312 h). Increasing the dose beyond 0.5 mg/kg at maximally responsive CTs (CT6 and CT15) resulted in progressively smaller but significant shifts. Thus, 0.5 mg/kg is the optimal dose in this species. The fact that risperidone resets the circadian rhythm in a mammal can be extended to clinical studies and used for optimal adjustment of the circadian rhythm in mental disorders. Conversely, risperidone administration for various treatments must be carefully timed to prevent unwanted phase shifts in patients.

Cinnamic acid shortens the period of the circadian clock in mice

Cinnamic acid (CA) derivatives have recently received focus due to their anticancer, antioxidant, and antidiabetic properties. The present study aimed to determine the effects of cinnamic acid on the circadian clock, which is a cell-autonomous endogenous system that generates circadian rhythms that govern the behavior and physiology of most organisms. Cinnamic acid significantly shortened the circadian period of PER2::LUC expression in neuronal cells that differentiated from neuronal progenitor cells derived from PER2::LUC mouse embryos. Cinnamic acid did not induce the transient mRNA expression of clock genes such as Per1 and Per2 in neuronal cells, but significantly shortened the half-life of PER2::LUC protein in neuronal cells incubated with actinomycin D, suggested that CA post-transcriptionally affects the molecular clock by decreasing Per2 mRNA stability. A continuous infusion of CA into mice via an Alzet osmotic pump under constant darkness significantly shortened the free-running period of wheel-running rhythms. These findings suggest that CA shortens the circadian period of the molecular clock in mammals.

Small Heterodimer Partner (NR0B2) Coordinates Nutrient Signaling and the Circadian Clock in Mice.

Circadian rhythm regulates multiple metabolic processes and in turn is readily entrained by feeding-fasting cycles. However, the molecular mechanisms by which the peripheral clock senses nutrition availability remain largely unknown. Bile acids are under circadian control and also increase postprandially, serving as regulators of the fed state in the liver. Here, we show that nuclear receptor Small Heterodimer Partner (SHP), a regulator of bile acid metabolism, impacts the endogenous peripheral clock by directly regulating Bmal1. Bmal1-dependent gene expression is altered in Shp knockout mice, and liver clock adaptation is delayed in Shp knockout mice upon restricted feeding. These results identify SHP as a potential mediator connecting nutrient signaling with the circadian clock.

The Zfhx3-Mediated Axis Regulates Sleep and Interval Timing in Mice

SummaryAn AT motif-dependent axis, modulated by the transcription factor Zfhx3, influences the circadian clock in mice. In particular, gain of function of Zfhx3 significantly shortens circadian rhythms and alters the transcriptional activity of an important class of neuropeptides that controls intercellular signaling in the suprachiasmatic nucleus (SCN) of the hypothalamus. The ZFHX3/AT axis revealed an important, largely cell-nonautonomous control of the circadian clock. Here, by studying the recently identified circadian mouse mutant Zfhx3Sci/+, we identify significant effects on sleep homeostasis, a phenomenon that is outside the canonical circadian clock system and that is modulated by the activity of those neuropeptides at a circuit level. We show that the Zfhx3Sci/+ mutation accelerates the circadian clock at both the hourly scale (i.e., advancing circadian rhythms) and the seconds-to-minutes scale (i.e., anticipating behavioral responses) in mice. The in vivo results are accompanied by a significant presence of sleep targets among protein-protein interactions of the Zfhx3Sci/+-dependent network.

RBP4 functions as a hepatokine in the regulation of glucose metabolism by the circadian clock in mice.

As one of the key adipokines, retinol binding protein 4 (RBP4) is suggested to positively correlate with insulin resistance; however, not all clinical studies support this association. Although some explanations are proposed for this discrepancy, the temporal aspect of RBP4 secretion has not been considered. Aryl hydrocarbon receptor nuclear translocator-like (also known as BMAL1) and its target D site-binding protein (DBP) are both pivotal transcription factors of the circadian core clock. Given the overwhelming presence of circadian control in metabolism and the principal role of the liver in RBP4 secretion, we hypothesised that RBP4 may oscillate under the control of BMAL1 and act as a hepatokine, participating in the maintenance of glucose homeostasis by the circadian clock. We used liver-specific Bmal1 (also known as Arntl)-knockout mice and recombinant adenoviruses expressing short-hairpin RNA (shRNA) specific for Dbp or Rbp4 in the liver. RBP4 displayed diurnal oscillations in the liver and plasma, which were dampened in liver-specific-Bmal1-knockout mice. BMAL1 regulated hepatic RBP4 expression via its direct target, DBP. Hepatic knockdown of RBP4 or DBP increased whole-body insulin sensitivity in mice in a time-of-day-dependent manner. Conversely, hepatic overexpression of RBP4 reversed the insulin-sensitising effects of liver-specific depletion of BMAL1. Our results not only provide a novel mechanism for circadian regulation of RBP4, but also unveil a critical role of RBP4, acting as a hepatokine in the regulation of glucose metabolism by the circadian clock.

Glucocorticoids Reset the Nasal Circadian Clock in Mice

The symptoms of allergic rhinitis show marked day-night changes that are likely to be under the control of the circadian clock, but the mechanism of this control is poorly understood. Because most peripheral tissues have endogenous circadian clocks, we examined the circadian rhythm of the clock gene product PERIOD2 (PER2) in the nasal mucosa of male mice using a luciferase reporter and demonstrated for the first time the phase-dependent effects of dexamethasone (DEX) on nasal PER2 rhythm in vivo and ex vivo. The phase shifts in PER2 rhythm caused by DEX were observed around the peak phase of serum glucocorticoids, suggesting that the circadian rhythm of endogenous glucocorticoids regulates the peripheral clock of the mouse nasal mucosa. From the viewpoint of circadian physiology, the best time to administer intranasal steroid treatment for allergic rhinitis would be when no phase shift is caused by DEX: in the early evening in diurnal humans.